Diverse thioether macrocyclized peptides through a radical SAM maturase

Disulfide bonds stabilize many bioactive peptides, but their susceptibility to reduction under physiological conditions limits broad applicability in biotechnology. PapB is a promiscuous radical <i>S</i>-adenosyl-L-methionine enzyme that is involved in the maturation of PapA, which is a ribosomally produced and posttranslationally modified polypeptide. PapB introduces six thioether linkages between internal Cys residues and carbon atom that is α to the side-chain carboxylate of Asp/Glu residues C-terminal to the Cys residues. Herein, we show that PapB also efficiently couples an internal Cys thiol to the C-terminal carboxylate of peptides terminating in D- or β-amino acids, forming α- or β-thioether macrocycles. Moreover, PapB tolerates β- and N-methyl amino acids within the peptide, resulting in the formation of macrocycles that are comprised entirely of unnatural amino acids, such as peptides containing all β-residues. These findings establish PapB as a sequence-agnostic thioether ligase for efficient C-terminal macrocyclization. Our work expands the enzymatic toolbox for constructing conformationally constrained peptides for therapeutics and chemical biology.